The human gut microbiota is dominated by the Bacteroidetes and Firmicutes divisions of Bacteria, and provides us with physiological traits we have not had to evolve on our own, including the ability to process otherwise indigestible dietary polysaccharides. This grant renewal focuses on characterizing the genomic and metabolomic features of the microbiota's keystone species, the operating principles that underlie their nutrient for aging/sharing behaviors, and the physiological benefits we accrue from our mutualistic (symbiotic) relationship with them. We have used germ-free (GF) mice colonized with Bacteroides thetaiotaomicron (B. theta), a saccharolytic anaerobe, to conduct the first reported genome-wide transcriptional profiling of a prominent human gut symbiont in vivo. Functional genomic and mass spectrometry-based metabolomic studies showed how B. theta's adaptive foraging behavior could help stabilize the microbiota in the face of changes in dietary polysaccharides. We now propose to systematically add complexity to this gnotobiotic mouse model to examine interactions between human-derived Bacteroidetes we have sequenced (B. theta, B. vulgatus, B. distasonis), Methanobrevibacter smithii (a dominant methanogenic archaeon in the human colon), and members of Firmicutes (Eubacterium rectale, E. eligens). Aim 1. Characterize the genomic and metabolic underpinnings of Bacteroidetes-Archaeal mutualism and its impact on host biology as follows: (i) produce a finished, annotated M. smithii genome (first Methanobrevibacter species to be sequenced); (ii) colonize GF mice fed standard polysaccharide-rich chow, a chow enriched for fructans, or a high fat Western diet with B. theta and/or M. smithii; (iii) perform whole-genome transcriptional profiling of both microbial species in vivo using custom GeneChips; (iv) simultaneously profile host transcriptional responses using laser capture microdissected colonic epithelium and mouse GeneChips; (v) perform in silica reconstructions of the metabolome based on the transcriptome datasets and use the results to direct hypothesis-based biochemical and genetic analyses of the contributions of microbial metabolic pathways to the mutualism; (vi) assess the generality of our findings using B. vulgatus and B. distasonis. Aim 2. Characterize the contributions of the Firmicutes to nutrient sharing in a simplified 3-component model of the human microbiota composed of a prominent member of this division, a member of Bacteroidetes, plus a methanogen. We will create an initial community by colonizing GF mice with E. rectale (a member of the human colonic microbiota whose genome we are currently sequencing), B. theta and M. smithii. Microbial and host transcriptional and metabolic responses will then be assessed as a function of community membership and diet. Based on the results, we will examine the properties of consortia containing selected combinations of Bacteroides spp., M. smithii, and E. rectale/E. eligens. These studies should provide new approaches for monitoring and manipulating the functions of the microbiota to promote human health.